Cardiomyocytes in adults have lost proliferating activity and the only way to treat diseases such as severe myocardial infarction and cardiomyopathy is heart transplantation. As of today, however, the shortage of heart donors still stands in the way and there is an urgent need for finding a therapeutic method other than heart transplantation. In contrast, preparing and purifying cardiomyocytes ex vivo and using them as a replacement of cardiomyocytes during treatment of disease is expected to be one of the most promising methods for saving patients with heart disease who have nothing to resort to except heart transplantation.
Cardiomyocytes are known to be obtainable by various methods including differentiation of stem cells (e.g. embryonic stem cells and a variety of adult stem cells) and acquisition from embryos. Depending on the pluripotent stem cell to be used, a suitable differentiation inhibiting factor (e.g. feeder cell or a leukemia inhibiting factor (LIF) in the case of using mouse pluripotent stem cells, or feeder cell, a basic fibroblast growth factor (bFGF) or a transforming growth factor (TGF) in the case of using human pluripotent stem cells) is removed from a culture medium to thereby induce formation of cell masses (embryoid bodies) and this is known as a method that can initiate differentiation of stem cells into cardiomyocytes.
The mode of ex vivo differentiation of stem cells into cardiomyocytes mimics some of the stages of in vivo physiological development and especially concerning events during early development, the modes of physiological development that takes place in fertilized egg cells and in vitro differentiation have a lot in common. The chronology of ex vivo differentiation into cardiomyocytes is the same as that of physiological development, starting with a differentiation of stem cells into undifferentiated mesodermal cells, some of which differentiate into programmed cardiomyocytes (pre-cardiac mesoderm) which in turn differentiate into cardiomyocytes.
Since pluripotent cells are cells that have the ability to differentiate into all cells that constitute an organ, it is technically difficult to differentiate them into cardiomyocytes only. It is also very difficult to ensure that all pluripotent stem cells are simultaneously induced to the differentiation stage, so it is quite common that stem cells remain undifferentiated in embryoid bodies.
Thus, an attempt to induce the differentiation of stem cells into cardiomyocytes ex vivo involves a problem deleterious to clinical application in that any types of stem cells can result in producing cells other than cardiomyocytes as a by-product or that some cells might remain undifferentiated. Especially, the residual undifferentiated cells have proliferating activity and are capable of differentiating into a great variety of cells, so if cells transplanted into the living body used in the therapy contain any residual undifferentiated cells, the likelihood that teratoma is formed from such undifferentiated cells is extremely high. For this reason, a cell population containing cardiomyocytes prepared by inducing the differentiation of pluripotent stem cells might be directly transplanted into the living body for treatment without great difficulty. Therefore, in order to ensure that a treatment using cardiomyocytes derived from pluripotent stem cells is performed with safety to secure an ideal therapeutic effect, it is necessary to find a method by which undifferentiated pluripotent stem cells are completely excluded and cardiomyocytes are highly purified (namely, a method for removing cells other than cardiomyocytes).
A currently known method for purifying cardiomyocytes is by preliminarily introducing a certain marker gene (e.g. GFP) into the genome of a stem cell (Non-Patent Document 1). However, this method requires genomic alteration, which itself presents an aesthetic problem and it also involves unpredictable serious risks in safety, such as a change in cell's canceration rate (Non-Patent Document 2). A method involving genomic alteration has also been reported as a way to positively remove undifferentiated pluripotent stem cells (Non-Patent Document 3). A method taking a different approach has been reported, in which ceramide analogues known to have a cell death inducing action are used to induce cell death in embryonic stem cells in a comparatively specific way (Non-Patent Document 4). However, this method does not assure satisfactory removal of pluripotent stem cells since the group of cells cultured after treatment with the ceramide analogues contained (OCT positive cells) in an amount as much as a third of those found in the untreated cell group (control). And as regards the removal of human embryonic stem cells, Non-Patent Document 4 only mentions that cells undergoing apoptosis were found and it does not say that satisfactory removal of pluripotent stem cells was effected. A method of using cytotoxic antibodies has been reported (Non-Patent Document 5) but the document states that, even after the treatment with the antibodies by this method, approximately 20% of embryonic stem cells still remained to be removed. As a further problem, utilization of the method involves several constraints such as the need to avoid the antigenicity of the antibodies before they can be used for therapeutic treatment. Thus, the known methods for inducing cell death have a room for improvement as a way to purify cardiomyocytes that can be used in the treatment of myocardial diseases, so it is desired to develop a new and even more efficient method for inducing cell death.